P821: HIGH-DIMENSIONAL SPECTRAL CYTOMETRY CHARACTERIZATION OF THE CIRCULATING IMMUNE CELL PROFILE IN ASYMPTOMATIC AND SYMPTOMATIC MYELOMA PATIENTS.
Authors: Joana Caetano, Filipa Barahona, Manuel Neves, Bruna Velosa Ferreira, Sónia Leocádio, Ana Queirós, Paulo Lúcio, Cristina João
Journal: HemaSphere (2023)

Topic: 13. Myeloma and other monoclonal gammopathies - Biology & Translational Research Background: Immune system dysfunction is a hallmark of multiple myeloma (MM), contributing to disease progression and therapy resistance. Efforts to understand the immune landscape in MM are essential but most studies detail the immune bone marrow environment niche, which implies an invasive approach. The use of liquid biopsies to assess patients’ peripheral cellular populations with high sensitivity is currently possible due to novel multi-parameter, single-cell analysis such as wide spectrum flow cytometry. This research may enlighten situations of immune suppression while revealing alterations associated with prognosis. Aims: The objective of this study is to widely characterize the profile of peripheral immune cells enabling the detection of relevant variations throughout myeloma disease stages. Methods: The cohort is composed of 5 healthy donors (HD 2 female; median age 70), 22 monoclonal gammopathy of undetermined significance (MGUS; 12 female; median age 68), 7 smoldering myeloma (SMM; 2 female; median age 70) and 12 MM (MM; 6 female; median age 63) patient. Peripheral blood samples were evaluated in a 5-laser Aurora spectral flow cytometer (Cytek) using a 25-antibody panel. Data and statistical analyses were performed in FCS Express 7 (De Novo Software). Results: Distribution and functional state of the circulating immune populations were different across myeloma disease stages. MGUS patients showed higher levels of circulating CD4 T cells with naïve and central memory (p<0.01) compared to SMM and MM. These cell subsets showed lower levels of CD127 and PD1, and higher expression of CD38. Overall, effector cells (early-like effector, early effector, and terminal effector) were also increased in MGUS. The expression of PD1 in these subsets was higher in MM patients compared to HD and MGUS (1.5x and 2x, respectively). In MM (both active and indolent) there was a higher frequency of regulatory T cells (1.5x) with higher expression levels of PD-1 and lower of CD38. Central memory CD8 T cells were significantly lower in MM patients (p<0.01) compared to other patient groups, as was expression of CD127 and PD1. MGUS showed higher frequencies of naïve, early-like and early effector CD8 T cells than MM. MM patients showed higher levels (1.5x) of terminal effector cells with CD28 expression. The naïve B cell subpopulation was increased in MGUS compared to SMM (1.5x) and overt MM (1.5x). Memory B cells were mostly IgD- in all patients, with the frequency of plasmablasts being higher in MM. Higher levels of both NK and NKT cells were identified in MM (p<0.01) compared to SMM or MGUS, with a less differentiated phenotype (early NK). Gamma-delta T cells were also increased (1.5x) in MM patients. Higher levels of dendritic cells were found in MM patients with the CD11c+CD16- subpopulation being more frequent. Classical monocytes were increased in MM, with lower expression levels of CD38. In general, the differences identified in the various immune cell populations’ abundance and activation status seem to indicate higher exhaustion and less naiveness phenotype in patients with active MM, reflecting the immunocompromised state of these patients related to MM previously to treatment start. Summary/Conclusion: These results suggest the potential application in routine practice of high-dimensional spectral immune profile as a biomarker for clinical monitoring. Further investigation is ongoing to increase the study cohort and address the clinical benefit of its use in a combined liquid biopsy approach and as a tool to informed- treatment selection.Keywords: Flow cytometry, Myeloma, liquid biopsy, Immunity